Multimodal Optical Imaging Platform for Studying Cellular Metabolism

Our research involves leveraging our multimodal microscope to measure molecular and metabolic differences in several pathologies and visualize their spatial heterogeneity. The multimodal approach to optical imaging enables us to identify pathophysiological changes from a variety of perspectives. The multimodal approach to optical microscopy is continuously expanding its applications, particularly in the clinical setting, where the development of micro endoscopes has opened an avenue for clinical imaging.

The current experimental challenges lie in the complexity of incorporating all the hardware with one another, which is part of the reason why we utilize a custom-built microscope system. Through the use of our multimodal optical imaging platform, we have made significant strides in label-free bioorthogonal disease study, including the classification of different breast cancer subtypes and the analysis of lipid metabolism in the drosophila and mouse brain. Using our label-free multimodal optical imaging, we're able to visualize the metabolism, morphology, and molecular composition simultaneously, which is a powerful tool for investigating diseases and the aging process.

To begin, warm up the laser and wait for approximately 15 to 20 minutes. Power on the control box, followed by touch panel controller, AC adapter for main laser remote, and AC adapter for sub laser remote, then power on the silicon photo diode detector and lock-in amplifier. Set up the laser system with a pump beam tunable from 780 nanometers to 990 nanometers, with a pulse width of five to six picoseconds and an 80 megahertz repetition rate.

The stokes laser beam should have a fixed wavelength of 1031 nanometers, with a six picoseconds pulse and an 80 megahertz repetition rate. Ensure both pump and stokes beams are at low power, at least 20 milliwatts to be visible on the alignment plate. Apply oil to the high numerical aperture oil condenser.

Mount the microscope slide onto the oil condenser, and place a large water droplet on the microscope slide for the 25X water objective. Adjust the Z stage to tune the focus until the bright brightfield image of the biological sample is visible under the 25X water objective. Begin the imaging process in the correct sequence to avoid photobleaching.

To switch between MPF and SHG quickly, switch from the pump beam to the fixed stokes beam. Select the image resolution as 512 by 512 pixels. Set the dwell time to eight microseconds per pixel for MPF and SHG, with an average frame above three.

Use 40 microseconds per pixel with an average frame of two for the SRS modality. To acquire autofluorescence with MPF, turn off the stokes laser beam. Tune the pump laser to 800 nanometers to excite NADH and flavin.

Acquire the collagen fiber signal using SHG. Turn off the pump laser beam and only use the stokes laser beam at a power of 500 milliwatts. Obtain the spatial distribution of proteins and lipids using SRS.

Keep both laser beams on and adjust the laser beam frequency to match the specific vibrational mode for each molecule. To acquire the SRS hyper-spectral image data sets, select suite mode and set the wavelength range from 781.3 nanometers to 806.5 nanometers. Choose a stack number of at least 60 and capture the hyper-spectral image stack.

Save all images of the same regions of interest in the same folder, and ensure the image format is Olympus OIR file. Autofluorescence and SRS imaging successfully captured metabolic and structural information from human lung tissue. Ratiometric analysis of the optical redox ratio and lipid unsaturation ratio provided spatial distributions of metabolic activity and molecular composition in human lung tissue.

Quantitative comparison of oxidative stress and lipid unsaturation between healthy and tumor tissue reveal differences in metabolic states.